To preserve the integrity of their genomic DNA, eukaryotic cells utilize a variety of surveillance mechanisms known as checkpoint controls. For example, during cell duplication, cells must make sure that they have replicated their DNA accurately. To cope with the challenges of copying the DNA precisely and rectifying any problems that arise in the process, cells utilize a myriad of regulatory proteins. In vertebrates, the kinase ATR acts as a pivotal regulator in the mechanisms that detect problems with DNA replication and enable cells to address such defects. A key feature of this kinase is that it undergoes precisely regulated activation upon genomic perturbation. Accordingly, this activation involves elaborate control mechanisms. For example, a binding partner known as ATRIP helps to recruit the ATR-ATRIP complex to RPA-coated regions of single-stranded DNA, which are characteristic of numerous detrimental DNA lesions. However, the ATR- ATRIP complex still remains weakly active upon associating with RPA on the DNA. At some point thereafter, ATR-ATRIP interacts with another protein called TopBP1. This binding results in a massive increase in the kinase activity of ATR and represents the culmination of numerous steps that trigger activation of a checkpoint response. Past studies have indicated that the Rad9-Hus1-Rad1 (9-1-1) complex plays a role in controlling the interaction of TopBP1 with ATR-ATRIP. However, many aspects of this overall process have remained nebulous. It has also been unclear whether activation of ATR occurs solely through this route in animal cells. We have recently observed that the Mre11-Rad50-Nbs1 (MRN) complex plays a novel role in the activation of ATR in response to aberrant DNA replication. The MRN complex had been best known for its role in another type of checkpoint mechanism, namely, the response to double-stranded DNA breaks (DSBs). In the upcoming grant period, a variety of studies will be carried out to elucidate the mechanism by which MRN collaborates with TopBP1 and other checkpoint control proteins to promote the activation of ATR. A systematic series of experiments will be conducted to explore: (1) how the nuclease activity of MRN affects checkpoint induction at replication forks; (2) how MRN and TopBP1 interact with one another and with replication forks during the checkpoint response; and (3) how these steps ultimately lead to the activation of ATR. Moreover, searches will be undertaken for novel regulators in these pathways. These studies will be performed with both Xenopus egg extracts and human tissue culture cells. This strategy will capitalize upon the complementary advantages of each experimental system. Overall, these studies will exploit new perspectives on checkpoint signaling and hence promise to uncover original insights into the mechanisms that safegaurd genomic integrity. This information would be invaluable for understanding how cells forestall cancer-promoting mutations and other disease-inducing genetic abnormalities.